Use of complex forms of calmodulin-like skin protein clsp

ABSTRACT

The invention also relates to the use of these complex forms for the purposes of screening for biological or chemical compounds capable of modulating a biological activity of said complex forms and/or for preparing and/or improving a pluristratified cell model.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/686,672filed Jan. 13, 2010, pending, and claims the benefit of U.S. applicationSer. No. 61/202,077 filed Jan. 27, 2009 and FR 0950162 filed Jan. 13,2009.

FIELD OF THE INVENTION

The present invention relates to the use of complex forms ofcalmodulin-like skin protein (CLSP), of peptide fragments of thisprotein or of analogues thereof, as a marker for evaluating a state ofchronological ageing of an epithelium, and in particular of theepidermis.

DESCRIPTION OF THE BACKGROUND

Epithelia are tissues of which the cells are joined to and interlinkedwith one another and lie on a basal membrane. They form either anexternal covering, for example at the surface of the skin, or theepidermis, or an internal covering, at the surface of a mucosa. They canalso form glands.

More specifically, these epithelia are structures of which thehomeostasis results from the implementation of a finely regulated set ofintracellular and extracellular signals acting at all the stages of cellproliferation, migration and differentiation, and also of the synthesisof the various extracellular matrix components. These signals can inparticular result from the action of factors produced by keratinocytes.

The maintaining of the correct physiological functions of an epitheliuminvolves, in particular, epithelial terminal differentiation and/orproteoglycan synthesis.

As regards more particularly the epidermis, it is an epithelium,conventionally divided up into a basal layer of keratinocytescontaining, in particular, skin stem cells and constituting thegerminative layer of the epidermis, a “spiny” layer constituted ofseveral layers of polyhedral cells placed on the basal layer, a“granular” layer comprising one to three layers said to be of flattenedcells containing distinct cytoplasmic inclusions, keratohyalin granules,and finally, a set of upper cells, called horny layer (or stratumcorneum) constituted of keratinocytes at the terminal stage of theirdifferentiation, called corneocytes.

The stratum corneum, the outermost part of the skin which performs thefunction of a barrier between the organism and the environment, and thehair shaft, the emerging part of the hair follicle which constitutes thehead of hair, both represent the result of the keratinocytedifferentiation process. Epidermal differentiation follows a process ofmaturation in which keratinocytes from the basal layer differentiate andmigrate so as to result in the formation of corneocytes, which arecompletely keratinised dead cells. This differentiation is the result ofperfectly coordinated phenomena which will result in the thickness ofthe epidermis being kept constant and thus ensure the homeostasis of theepidermis.

Many skin disorders or pathological conditions can result from adysfunction of epidermal homeostasis.

Thus, in the case of aged skin, this dysfunction is generally manifestedthrough the appearance of wrinkles (microrelief and deep wrinkles), aloss of elasticity, a rough feel and dryness. From the histologicalpoint of view, a flattening of the dermo-epidermal junction and adecrease in thickness of the dermis and of the epidermis are observed.The collagen and glycosaminoglycan content decreases. The barrierfunction of the skin is impaired. All these phenomena are increased bychronic exposure to the sun.

Similarly, this dysfunction may be worsened in women during themenopause.

SUMMARY OF THE INVENTION

The present invention results more particularly from thecharacterization by the inventors in the stratum corneum, of complexforms of the CLSP protein, the expression of which is found to bemodulated differently over the course of the chronobiological ageing ofan epithelium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: it illustrates the relationship between age and the amount ofcomplex forms according to the invention present in the skin of theforearm and of the face.

FIG. 2: it illustrates the effects linked to the application of acosmetic composition, composed of 10% of bifidiobiotic (CLR complex) and0.002% of phytosphingosine-SLC (treatment “G”), on the amount of complexforms according to the invention, in the skin of the forearm and of theface.

DETAILED DESCRIPTION OF THE INVENTION

CLSP is a calcium-binding protein belonging to the calmodulin family,the precursor form of which comprises 146 amino acids. It has amolecular weight of approximately 16 kDa and an amino acid sequencehomology of approximately 52% with calmodulin.

The structure of CLSP comprises 4 potential “EF-hand” calcium-bindingsites, potential N-glycosylation sites, multiple potentialphosphorylation sites and a myristylation site.

The binding of calcium to CLSP induces a change in its conformationwhich leads to its activation, enabling in particular its interactionwith various target proteins.

An increase in the mRNA encoding CLSP has been shown over the course ofphoto-induced ageing (Urschitz, J. et al., J. Invest. Dermatol., 2002,119:3-13) i.e. ageing resulting from repeated exposure of the skin torays from the sun, a process distinct from that of chronological ageing,i.e. of physiological ageing linked to the passing of time.

For its part, document US 2004/0142335 observes, by means of atranscriptome analysis, an increase in the expression of the mRNAencoding the CLSP protein in skin of an aged individual compared withthat of a young individual.

Thus, CLSP could in particular be involved in keratinocyte terminaldifferentiation (B. Mehul et al., J. Biol. Chem., 2000, April 28;275(17):12841-7), (EP 1 204 744).

More recently, the inventors have, unexpectedly, noted a decrease in theexpression of particular forms of CLSP in the stratum corneum of agedhuman epidermis. Quite clearly, the forms characterized by the inventorscorrespond to stages of maturation of the protein which are distinctfrom those of the forms of CLSP considered up until now and inparticular mentioned above.

As emerges from what follows, the complex forms have been characterizedby the inventors by means of the implementation of a specific ELISAassay technique, in particular described in Example 1 hereinafter.

Thus, according to one of its aspects, the present invention relates tothe use of at least one complex form of calmodulin-like skin protein, orCLSP, which derives at least in part from a polypeptide having an aminoacid sequence encoded by a nucleic acid sequence represented completelyor partly by SEQ ID NO 1, or an analogue thereof, or a fragment thereof,as a tool for the characterization, in particular in vitro or ex vivo,of a state of chronological ageing of an epithelium, and in particularof an epidermis.

Accordingly, the present invention relates to a method for the in vitroor ex vivo characterization of a state of chronological ageing of anepithelium comprising a step of determining at least one complex form ofCLSP according to the invention, within said epithelium.

By “chronological ageing”, within the meaning of the invention, it isintended to set aside from the scope of the invention the cutaneousphenomena which are consecutive to sun or U.V. exposure, namely thephoto-induced skin ageing.

For the purpose of the present invention, the term “complex form” isintended to cover any conjugate of the CLSP protein or derivativethereof, with either another protein distinct from CLSP or a fragmentthereof, or another CLSP protein or a fragment thereof.

For the purpose of the present invention, the term “derivative of theCLSP protein” denotes a fragment thereof or an analogue as definedhereinafter.

More specifically, a complex form may derive from the association of thewhole CLSP protein or a fragment thereof, with either itself, or one ofits fragments, or else with a target protein, or even with a proteinstructure such as a cornified envelope that has been proteolysed in thestratum corneum.

Thus, according to a first variant, the complex forms underconsideration according to the invention may be dimer forms of the CLSPprotein, or else the conjugate of two identical or different peptidefragments of this protein.

According to another variant, these complex forms may be the productresulting from the association of the CLSP protein or a fragmentthereof, with a secondary target protein. By way of illustration ofthese target proteins capable of interacting with CLSP and/or a fragmentthereof, mention may be made in particular of the 14.3.3 betapolypeptide, 14.3.3 sigma, annexins II and V, the calreticulins, ERp72,nucleolin, transglutaminase 3, MAGED1 (melanoma antigen gene D1), PPP4C(protein phosphatase 4) and USF2 (upstream transcription factor 2). Theymay also be peptide motifs such as the YWHAQ motif (14.3.3 tau).

Against all expectations, the inventors have noted that thequantification of these complex forms in the stratum corneum constitutesan effective and reliable means for defining the state of thecorresponding epidermis. As specified above, a decrease in theexpression of these complex forms is observed over the course ofchronological ageing.

In this respect, a decrease in complex form(s) in accordance with theinvention is indicative of a state of chronological ageing of anepithelium.

The characterization of these complex forms is therefore found to be ofparticular use for establishing a diagnosis of the state of anepidermis.

With regard to this diagnosis, it will be advantageously possible tooffer the individual a cosmetic treatment that is most particularlysuited to the profile of said individual. For obvious reasons, this typeof personalization in terms of cosmetic treatment is particularlydesired and liked by users. The present invention makes it possible toadvantageously meet this demand.

According to another of its aspects, the present invention relates to anoninvasive, in particular cosmetic, method for characterizing, inparticular in vitro or ex vivo, the chronological state of an epidermis,comprising at least the qualitative or quantitative characterization, inthe stratum corneum of said epidermis, of the expression of at least onecomplex form of CLSP according to the invention.

According to one variant embodiment, the piece of data or value obtainedvia such a method may be assessed in comparison to a reference piece ofdata or value, obtained, for example, from at least one epithelium, inparticular an epidermis, distinct from that which is the subject of thecharacterization, and the state of which is known.

According to one of its aspects, the present invention relates to amethod for characterizing the chronological state of an epidermis asdefined above, in which a decrease in complex forms in accordance withthe invention is indicative of a state of chronological ageing.

According to another of its aspects, the present invention is alsodirected towards a noninvasive, in particular cosmetic, method forcharacterizing, in particular in vitro or ex vivo, the effectiveness ofa cosmetic or therapeutic treatment intended to prevent and/or treat thesigns of skin ageing linked to chronological ageing in an individual,such as wrinkles and fine lines, comprising at least the qualitative orquantitative characterization of at least one complex form of CLSPaccording to the invention.

The expression “signs of skin ageing” is intended to mean all themodifications of the external appearance of the skin due tochronological ageing, for instance wrinkles and fine lines, wizenedskin, lack of elasticity and/or of tonicity of the skin, thinning of thedermis and/or degradation of the collagen fibres thereby leading to theappearance of slack and wrinkled skin.

The signs of skin ageing considered in the invention are thoseassociated with or resulting from chronological skin ageing.

In particular, the signs of skin ageing considered in the invention aredistinct from those associated with or resulting from the photo-inducedskin ageing.

By “photo-induced ageing”, it is intended to mean, in particular, theoxidative damages resulting from U.V exposure of the epidermis, and moreparticularly from U.V.A. exposure, resulting in the generation of freeradicals, the glycation of proteins or the excessive activation of somecell surface receptors, and on tissue level, the thickening of elastinfibers.

Thus, the present invention is directed to the prevention and/or thetreatment of the cutaneous manifestations associated with chronologicalskin ageing, and more particularly not associated with U.V. exposure.

The instant invention is more particularly directed to chronologicalskin ageing manifestations resulting from cell attrition and cellsenescence.

Also, chronological ageing, on epidermis cells level, manifests itselfby telomeres reduction, a decrease of cells stimulation due to hormonesdeficiencies, such as DHEA, or growth factors deficiencies, or due to adecrease in cell receptors numbers, and, on tissue level, notably byelastin fibers disappearance.

Such phenomena are not associated with an U.V. exposure, and, inconsequence, are not associated with photo-induced skin ageing.

Cellular and tissue deficiencies subsequent to chronological skin ageingmay result in xerosis, epidermis laxity, wrinkles and/or fine lines,cutaneous slackness, seborrheic keratose or cherry angioma.

More specifically, the method as described above may comprise at leastthe steps consisting in:

i. providing at least a first cutaneous surface sample representative ofsaid individual,

ii. quantifying, in said sample, especially via an ELISA assaytechnique, and in particular that described in Example 1 hereinafter, atleast one complex form according to the invention,

iii. repeating steps i. and ii. on a second skin surface samplerepresentative of said individual, and

iv. comparing the results obtained at the end of steps ii. and iii., inparticular in order to deduce therefrom information relating to at leastone effect of the treatment, said first and second skin surface samplescorresponding to different treatment stages.

The reference value or piece of data in step ii. may be a piece of dataobtained from the epithelium, in particular from the epidermis,representative of the individual who is the subject of the treatment,prior to the administration of said treatment or within a shorterchronological time in relation to the treatment start date.

According to one preferred embodiment, the first sample isrepresentative of a pretreatment state and the second is representativeof a state during the course of treatment or a post-treatment state.

According to one variant embodiment, the present invention relates toone of the methods above, in which an increase in complex form(s) isindicative of said treatment of the signs of skin ageing beingeffective.

According to one variant embodiment, one of the methods above may alsocomprise, at the end of the step of characterizing the complex forms ofCLSP, at least one additional step aimed at administering, to saidindividual, a care composition, in particular a cosmetic carecomposition, established or selected with regard to the informationobtained on said complex forms. This additional step may be consecutiveto the characterization step.

According to one embodiment, this composition may also be selected froma range of compositions, each one of the latter being more particularlysuitable for a type of information that may be obtained according to themethod of the invention.

Thus, the advantage of the present invention is to propose a simple andrapid method, firstly for establishing the characterization of thephysiological state of an epithelium, in particular of the epidermis ofthe skin and, secondly, for adjusting accordingly a treatment suitablefor said epithelium or epidermis of the skin.

The methods of the invention may be carried out in vitro, ex vivo or invivo.

As emerges from the description which follows, the methods according tothe invention are particularly advantageous insofar as theimplementation thereof does not require an invasive procedure.

This is because the localization, by the inventors, of these newbiomarkers for ageing in the stratum corneum makes it possible toquantitatively or qualitatively characterize the expression of saidmarkers by simple topical sampling.

Advantageously, this method may be carried out on a sample of stratumcorneum of the individual under consideration, simply taken bystripping. The sampling method may, for example, be a strippingtechnique consisting in applying a portion of adhesive tape to theepithelium under consideration, such as an epidermis. On detaching thisadhesive tape, a fraction of the epithelium, for example an epidermalfraction, is sampled. After protein extraction, said fraction is thenanalysed by means of an ELISA assay as considered in the presentinvention.

According to yet another aspect, the present invention also relates tothe use of at least one complex form in accordance with the invention,as a tool for screening for biological or chemical compounds capable ofmodulating, and in particular of promoting, the increase in and/or thestability and/or the biological activity of these complex forms.

Accordingly, the present invention relates to a method for screening forbiological or chemical compound capable of modulating the increase inand/or the stability and/or the biological activity of at least onecomplex form of CLSP according to the invention, comprising a step ofcontacting at least said complex form with at least one biological orchemical compound to be screened.

In particular, it relates to a method of screening for anti-ageingactive agents, comprising at least the steps consisting in:

a) bringing at least one cell type containing at least one complex formin accordance with the invention into contact with at least one testchemical or biological compound, under conditions suitable for amanifestation of the expression of said form, and

b) determining a content of at least one of said complex forms,

c) comparing said content determined in step b) with a content of saidcomplex form determined in the absence of test chemical or biologicalcompound.

According to one variant embodiment, the present invention relates to amethod as defined above, in which an increase in complex form(s) inaccordance with the invention is indicative of an active agent withanti-ageing properties.

Thus, advantageously, a step of selecting the active agent(s) for whichan increase in the content of complex form(s) in accordance with theinvention is observed may further be carried out, at the end of step c).

According to yet a final aspect, the present invention relates to thecosmetic use of an effective amount of complex form(s) in accordancewith the invention, as an agent useful for prevent and/or treat thesigns of skin ageing.

According to yet another aspect, the present invention relates to theuse of an effective amount of complex form(s) in accordance with theinvention for preventing and/or treating thinning of an epidermis and/ora loss of firmness, of elasticity, of density, and/or of tonicity of anepidermis and/or the formation of wrinkles and fine lines.

According to yet another aspect, the present invention relates to theuse of an effective amount of complex form(s) in accordance with theinvention or of at least one agent which modulates the formation of saidcomplex form(s), for preparing and/or improving a pluristratified cellmodel, in particular a reconstructed skin model.

According to yet another aspect, the present invention relates to amethod of preparing an isolated reconstructed skin, comprising at leastthe step consisting in bringing at least one complex form according tothe invention into contact with cells capable of generating an isolatedreconstructed skin, and in particular keratinocytes.

Definition of “Complex Forms” of CLSP

As specified above, the complex forms in accordance with the inventionderive at least in part from a polypeptide having an amino acid sequenceencoded by a nucleic acid sequence represented completely or partly bySEQ ID NO 1, or an analogue thereof or a fragment thereof.

A complex form of CLSP may be in particular a multimer resulting fromthe association of at least the whole CLSP protein or a fragment thereofwith, either itself, or one fragment thereof, or yet with at least onetarget protein, even with at least one protein structure such asproteolysed cornified envelope in the stratum corneum.

The CLSP fragments according to the invention are fragments having alength and an activity suitable for their implementation as a complexform according to the invention.

More particularly, a complex form of CLSP may be a dimer, and moreparticularly a whole CLSP homodimer or a fragment thereof.

According to a particular embodiment, a dimer according to the inventionmay comprise a first monomer represented by the whole CLSP protein or afragment thereof, and a second monomer represented by a structuredistinct from the whole CLSP protein or a fragment thereof.

As examples of monomer represented by a structure distinct from thewhole CLSP protein or a fragment thereof suitable for the invention maybe mentioned the above-cited proteins and fragments thereof which areable to dimerize with the whole CLSP or a fragment thereof.

According to another particular embodiment, a complex form suitable forthe invention may comprise a peptide derived from the CLSP and distinctfrom the whole CLSP sequence, in particular distinct from the aminoacids sequence encoded by a nucleic acid sequence represented by SEQ IDNO 1.

In particular, a peptide derived from the CLSP suitable for theinvention may be a fragment of CLSP, and more particularly a fragment ofthe protein encoded by nucleic acids sequence represented by SEQ ID NO1, comprising an appropriate length and an activity suitable for its useas a complex form according to the invention.

For the purpose of the present invention, the expression “fragment of anucleic acid sequence” is intended to denote a nucleic acid sequencepartly encoding the complex forms in accordance with the invention, oran analogue thereof, and in particular a nucleic acid sequencerepresented by SEQ ID NO 1 or an analogue thereof.

The expression “analogue of a nucleic acid sequence” is intended todenote any nucleic acid sequence, optionally resulting from thedegeneracy of the nucleic acid code, and encoding at least a part of thecomplex forms having a sequence identical or analogous to that of thepolypeptide encoded by said nucleic acid sequence.

The nucleic acid sequences may be derived from any possible origins,i.e. either animal, in particular mammalian, and even more particularlyhuman or plant, or from microorganisms (viruses, phages, bacteria interalia) or else from fungi, without prejudging the fact that they may ormay not be present naturally in said organism of origin.

According to one variant embodiment, the complex forms according to theinvention may be soluble.

For the purpose of the present invention, the term “soluble” is intendedto describe the ability of the complexed peptide form underconsideration according to the invention to solubilize in water or in anaqueous medium without protein-denaturing substances of the chaotropicagent or ionic detergent type, for example, as opposed to the nativeCLSP, which can be extracted only in the presence of such agents.

According to another embodiment, the complex forms in accordance withthe invention derive at least in part from a polypeptide of which theamino acid sequence is represented completely or partly by SEQ ID NO 2,or an analogue thereof, or a fragment thereof.

For the purpose of the present invention, the term “CLSP” is intended todenote, in general, unless otherwise indicated, the sequence (SEQ ID NO2) of the protein which may or may not have undergone post-translationalmodifications of glycosylation type on the asparagine residues inposition 25 and/or 43, of phosphorylation type and/or of myristylationtype, capable of modifying its apparent molecular weight or itsisoelectric point.

It is, moreover, known that the primary sequence of a polypeptide, i.e.the series of its amino acids, determines sites specifically recognizedby protease enzymes, such as trypsin which, once the recognition ofthese sites has become effective, will induce cleavage of thepolypeptide by proteolysis. This proteolysis results in the generationof various peptides, or proteolytic fragments, of the complex forms ofCLSP.

The inventors have detected the presence of such peptides in the stratumcorneum.

Consequently, the invention extends to the complex forms comprising atleast a proteolytic fragment of CLSP having the peptide sequence SEQ IDNO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8,SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ IDNO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO24 and SEQ ID NO 25.

Thus, according to one particular embodiment, the complex forms inaccordance with the invention derive at least from a polypeptide ofwhich the amino acid sequence is represented completely or partly by anamino acid sequence chosen from SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5,SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ IDNO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24 and SEQ ID NO 25, andmixtures thereof.

The term “analogue of a polypeptide” is intended to denote anypolypeptide exhibiting a sequence homology, in particular with respectto one of the characteristic sequences of said polypeptide, and also abiological activity of the same nature.

This analogue may be a peptidomimetic agent.

The homology may be at least 85%, for example at least 90%, and forexample at least 95%. The homology may be determined by visualcomparison or by means of any computer tool generally used in the field,such as the BLAST programs available at www.ncbi.nlm.nih.gov and usedwith the default parameters.

The sequence homology may result from modifications derived frommutation or variation in the sequences of the peptides according to theinvention, originating either from the deletion of one or more aminoacids, or from insertion of one or more amino acids, or from thesubstitution of one or more amino acids in the characteristic sequencesof a polypeptide according to the invention.

For the purpose of the invention, the term “fragment” is intended todenote any peptide portion comprising at least 4, at least 6, inparticular at least 8, and more particularly at least 12 consecutiveamino acids of the CLSP protein, and a substantially similar biologicalactivity.

In general, the polypeptide analogues may comprise conservativesubstitutions with respect to the natural amino acid sequence.

Several of these modifications may be combined.

By way of example of mutations that may be considered in the presentinvention, mention may be made, non-exhaustively, of the replacement ofone or more amino acid residues with amino acid residues having asimilar hydropathic index, without however substantially affecting thebiological properties of the complex forms according to the invention.

The hydropathic index is an index assigned to amino acids according totheir hydrophobicity and their charge (Kyte et al. (1982), J. Mol.Biol., 157: 105).

The complex forms also covered by the present invention may derive frompolypeptides as defined above having undergone one or morepost-translational modification(s).

The term “post-translational modification(s)” is intended to encompassall the modifications that a peptide or a protein is capable ofundergoing at the end of its synthesis in a cell, such as, for example,one or more phosphorylation(s), one or more thiolation(s), one or moreacetylation(s), one or more glycosylation(s), one or more lipidation(s),such as a myristylation, a structural rearrangement such as theformation of disulphide bridges and/or cleavage within the peptidesequence.

The analogue has, moreover, substantially the same biological activityas the natural polypeptide.

According to one embodiment, the complex forms suitable for theimplementation of the invention may also derive from natural orsynthetic polypeptides, where appropriate capable of being obtainedafter enzymatic or chemical lysis of the native form of CLSP or bychemical or biological synthesis or by extraction from a biologicaltissue, for instance the skin, naturally expressing these complex forms,and also the various post-translational forms thereof.

Those skilled in the art can obtain the complex forms in accordance withthe invention by means of recombinant DNA based methods, for instancethose described in the manual “Molecular Cloning—A Laboratory Manual”(2nd edition), Sambrook et al., 1989, Vol. I-III, Coldspring HarborLaboratory, Coldspring Harbor Press, NY, (Sambrook).

According to another embodiment, the complex forms suitable for theimple-mentation of the invention may also be fused with anotherpolypeptide distinct from those identified above, a hydrophilic orhydrophobic targeting agent, a bioconversion precursor, or aluminescent, radioactive or colorimetric labelling agent.

In a non-limiting manner, mention may be made, as an example ofcompounds that can be coupled with the complex forms in accordance withthe invention, of fluorescent proteins such as Green FluorescentProtein, fluorescent chemical compounds such as rhodamine, fluorescein,or Texas Red®, phosphorescent compounds, radioactive elements, such as³H, ¹⁴C,³⁵S,¹²I or ¹²⁵I, or colorimetric labelling agents such aschromogenic substrates sensitive to the action of galactosidase, ofperoxidase, of chloramphenicol acetyltransferase, of luciferase or ofalkaline phosphatase.

Depending on the nature of the compounds that can be coupled with thecomplex forms in accordance with the invention, the coupling may beperformed by chemical methods, in particular by means of reactivechemical functions, or by molecular biology methods known to thoseskilled in the art.

By way of methods for detecting a polypeptide, mention may be made ofWestern blotting, slot blotting, dot blotting, ELISA (Enzyme LinkedImmuno-Sorbent Assay) methods of singleplex or multiplex type,proteomics or glycomics methods, staining of polypeptides in apolyacrylamide gel with a silver-based stain, with Coomassie blue orwith SYPRO, immunofluorescence, UV absorption, immunohistochemicalmethods in conventional, electron or confocal microscopy, FRET(fluorescence resonance energy transfer), TR-FRET (time-resolved FRET)methods, FLIM (fluorescence lifetime imaging microscopy) methods, FSPIM(fluorescence spectral imaging microscopy) methods, FRAP (fluorescencerecovery after photobleaching) methods, reporter-gene methods, AFM(atomic force microscopy) methods, surface plasmon resonance methods,microcalorimetry methods, flow cytometry methods, biosensor methods,radioimmunoassay (RIA) methods, isoelectric focusing methods, andenzymatic assays, methods using peptide chips, sugar chips, antibodychips, mass spectrometry methods, and SELDI-TOF spectrometry methods(Ciphergen).

As emerges from what follows, the detection of the complex formsaccording to the invention is favoured according to an ELISA method.

Use of the Complex Forms According to the Invention, for the Purposes ofDiagnosing a State of the Epidermis and/or of Screening for Anti-AgeingActive Agents

As specified above, according to another of its aspects, the presentinvention relates to non-invasive methods for characterizing, inparticular in an in vitro or ex vivo manner, the surface state of anonpathological epidermis or alternatively the effectiveness of acosmetic or therapeutic treatment, aimed at qualitatively orquantitatively characterizing the expression of the complex forms formedat least in part of CLSP, or from a derivative or fragment thereof.

These methods are particularly advantageous insofar as theimplementation thereof does not require obligatory recourse to asurgical technique in order to carry out such a characterization.

The methods according to the invention, described hereinafter, can becarried out on a sample, for example an isolated sample, of epithelium,and in particular of epidermis, taken from an individual.

The methods according to the invention may also be carried out on asample of epithelium, and in particular of epidermis, taken from anepithelial cell model, and in particular an epidermal cell model, orfrom a reconstructed isolated skin in order to define the state thereof.

An extract of the epidermis can thus be obtained by simple stripping anddirectly analysed by the ELISA method as described in Example 1hereinafter.

The stripping technique consists in applying a sticky surface to thesurface of the epidermis, such as Blenderm® from 3M, D′squam (commercialadhesive from CuDERM), or cyanoacrylate glue. By virtue of thesestrippings, the adherent corneocytes and the content of theirintercellular spaces can be sampled and subsequently subjected to anextraction which makes it possible to access the protein content.

The taking of a sample suitable for the method may also be carried outmore directly by “washing” the skin surface by means of, for example,accessories of the vane turbine type or of the spiral cell type, asdescribed in patent FR 2 667 778 combined with a fluid circuit, orsimply by addition/removal of a drop of buffer at the surface of theskin.

By way of indication of other sampling methods suitable for implementingthe invention, mention may be made of methods based on scraping theupper part of the stratum corneum by means of a twin blade system or byshave biopsy. This technique makes it possible to collect squamae whichcan then be analysed directly by various techniques in order todetermine the mineral, amino acid or lipid contents.

At the end of the sampling, the sample is characterized by the ELISAmethod.

This method is based in particular on the use of antibodies suitable forthe detection of complex forms under consideration according to theinvention. An antibody capable of being used as a tool for evaluating astate of an epidermis may be obtained by any method known to thoseskilled in the art, as described in “Antibodies: A Laboratory Manual”,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).

As described in Example 1 which follows, the method of detecting thecomplex forms of CLSP requires in particular the use of two types ofmonoclonal antibodies, developed by the company covalAb, i.e. a captureantibody (clone DB15C9) and a selection antibody labelled with“sulpho-tag” (CLSP clone DB7g12).

Method for the Diagnosis of a State of the Epidermis

According to one embodiment, a method for characterizing a state of anepithelium, for example an epidermis, comprises at least the stepsconsisting in:

a) determining, in a surface sample of said epithelium, a content of atleast one complex form in accordance with the invention, and

b) comparing said content determined in step a) with a reference value.

Advantageously, a method of the invention is noninvasive.

A reference value may, for example, be a content of complex forms,determined on a sample of epidermis taken from an epithelium, and inparticular normal skin, i.e. skin which is satisfactory from thephysiological point of view, like, for example, young skin.

The measurement of a reference value may be carried out in parallel orsequentially to the determination of said content of said complex forms.

Comparison of a determined content with a reference value may make itpossible to evaluate a deviation relative to this value.

The analysis of the size and/or of the nature of this deviation(negative or positive) may be informative with regard to the state ofthe epidermis.

The characterization of a state of an epidermis may be indicative of apossible skin disorder which may be corrected through the use ofcompounds capable of modulating the expression of the complex formsunder consideration in the invention.

Thus, a decrease in complex form(s) of the invention is indicative of astate of chronological ageing.

According to one embodiment, a method according to the invention may beimplemented in a method for the in vivo, in vitro or ex vivo diagnosisof a physiological state of an epithelium, and in particular of theepidermis, with a view in particular to assessing its state ofchronobiological ageing and, for example, its associated deficienciesand/or alterations in an individual.

Screening for Biological or Chemical Compounds

The present invention relates to the use of the complex forms inaccordance with the invention, as a tool for screening for biological orchemical compounds capable of modulating an increase in and/or thestability and/or the biological activity of these complex forms.

The present invention also relates to a method of screening forbiological or chemical compounds, or even anti-ageing active agents, orphysicochemical factors, capable of modulating the amount of the complexforms in accordance with the invention, comprising at least the stepsconsisting in:

a) bringing at least one cell type containing at least one complex formin accordance with the invention into contact with at least one testchemical or biological compound, under conditions suitable for therelease of said complex form,

b) determining a content of said complex form, and

c) comparing said content determined in step b) with a content of saidcomplex form determined in the absence of test chemical or biologicalcompound.

The comparison carried out in step c) may make it possible to deduceinformation regarding the property of said tested compound of modulatingthe expression of the complex forms in accordance with the invention.

As specified above, this method may also comprise a step aimed atretaining the active agents for which an increase in the content ofcomplex form(s) is observed.

More particularly, the method that is of use for characterizing theeffectiveness of a treatment capable of preventing and/or treating thesigns of skin ageing in an individual, may comprise at least the stepsconsisting in:

i. providing at least a first skin surface sample representative of saidindividual,

ii. quantifying, in said sample, especially via the ELISA assaytechnique disclosed in Example 1, at least one complex form underconsideration in the present invention,

iii. repeating steps i. and ii. on a second skin surface samplerepresentative of said individual, and

iv. comparing the results obtained at the end of steps ii. and iii., inparticular in order to deduce therefrom information relating to at leastone effect of the treatment, said first and second skin surface samplescorresponding to different treatment stages.

In the event that a reference value measurement is carried out prior tothe use of the test biological or chemical compound or of the testphysicochemical factor, the method according to the invention may alsomake it possible, where appropriate, to assess the potentialeffectiveness of said compound.

This amount may not be affected by the presence of said compound or onthe other hand, may be inhibited or stimulated.

In the event that an increase is observed, the compound tested iscapable of being used, for example, as an anti-ageing active agent.

According to one variant embodiment, the methods as defined above mayalso comprise at least one step consisting of administering to anindividual, a cosmetic care composition established or selected withregard to the information obtained at the end of step iv.

The determination of a content of the complex forms in accordance withthe invention is carried out by means of an ELISA method, in particularthat specifically described in Example 1 hereinafter.

According to another of the aspects of the present invention, thecomplex forms of CLSP as described above may be used in a cosmetic ortherapeutic composition.

It is understood that all the cosmetic or therapeutic compositions underconsideration according to the invention use a physiologicallyacceptable medium.

For the purpose of the present invention, the term “physiologicallyacceptable medium” is intended to denote a medium that is suitable forapplication of a composition to an epithelium or a keratin material,such as the skin, the scalp, the lips, the mucous membranes and keratinfibres such as the hair, the nails and body hair or, where appropriate,orally or parenterally.

For the purpose of the invention, the term “cosmetic” is intended todenote a use intended, principally, to provide an aesthetic and/orcomfort effect.

For the purpose of the present invention, the term “therapeutic” isintended to denote a composition that can be used in the context of aprophylactic and/or curative treatment, or of a method for evaluating astate of an epithelium, and in particular of the epidermis.

For the purpose of the invention, the term “prophylactic” or“preventive” is intended to mean the decreased risk of occurrence of aphenomenon, for example a pathological condition.

According to another embodiment, a cosmetic or therapeutic compositionin accordance with the invention may also comprise at least one cosmeticand/or therapeutic active agent.

As examples of active agents that can be used in the context of thepresent invention, mention may be made of cosmetic oils, such assilicone oils, plant oils of triglyceride type, hydrocarbon-based oilssuch as parleam oil and esters of fatty acids and of fatty alcohols.

Other active agents which make it possible to improve the state of theskin may also be used, such as moisturizing active agents, or activeagents which make it possible to improve the natural lipid barrier, suchas ceramides, cholesterol sulphates and/or fatty acids and mixturesthereof.

It may also be possible to use enzymes which have an activity on theskin, such as proteases, lipases, glucosidases, amidases, cerebrosidasesand/or melanases, and mixtures thereof.

In general, any composition of the invention may be applied to the skin(on any cutaneous region of the body) or to the mucous membranes(buccal, jugal, gingival, genital, conjunctival, etc.).

In a known manner, a cosmetic composition may also contain adjuvantswhich are customary in the cosmetics field, such as hydrophilic orlipophilic gelling agents, hydrophilic or lipophilic additives,preservatives, antioxidants, solvents, fragrances, fillers, screeningagents, odour absorbers and dyestuffs.

The amounts of the various constituents of the compositions according tothe invention are those conventionally used in the fields underconsideration.

The amount of complex form(s) in accordance with the invention,contained in a composition according to the invention, also referred toas “effective amount” of course depends on the nature of the compoundand on the desired effect, and may therefore vary to a large extent.

To give an order of magnitude, a composition may contain a complex formaccording to the invention in an amount representing from 0.00001% to50% of the total weight of the composition, in particular in an amountrepresenting from 0.001% to 10% of the total weight of the composition,and more particularly in an amount representing from 0.1% to 1% of thetotal weight of the composition.

A composition according to the invention may be more particularlyintended for reducing and/or treating conditions that may causedeterioration of the state of an epithelium, and in particular of anepidermis.

Such a state may be of chronological origin (i.e. linked to the timethat has passed, such as skin ageing) and/or indicative of a skindisorder, linked for example to photoageing.

Thus, a composition in accordance with the invention, especially acosmetic composition, may in particular be for use in preventing and/ortreating thinning of an epidermis and/or a loss of firmness, ofelasticity, of density and/or of tonicity of an epidermis and/or theformation of wrinkles and fine lines.

According to another embodiment, a composition in accordance with theinvention, in particular a cosmetic composition, may, especially, be foruse in preventing and/or treating cutaneous sign of dryness, inparticular in preventing and/or treating dehydration of an epidermis.

According to another embodiment, a composition in accordance with theinvention, especially a cosmetic composition, may be for use inpreventing and/or treating signs of epidermal ageing.

According to another aspect, the present invention relates to a methodfor the cosmetic treatment of the signs of skin ageing, comprising atleast one step consisting in applying at least one cosmetic compositionin accordance with the invention to at least a part of the skin, themucous membranes and/or the keratin fibres.

According to yet another aspect, the invention also relates to the use,in particular cosmetic and/or therapeutic use, of complex forms of theCLSP protein, of peptide fragments of this protein or of analoguesthereof, or of an agent which modulates the activity, the expressionand/or the stability of such a polypeptide, in particular for preventingthe signs of skin ageing, in particular for preventing and/or treatingaged skin.

The present invention also relates to the use of an effective amount ofcomplex forms in accordance with the invention for preparing and/orimproving a pluristratified cell model, especially of epidermal ormucosal type, and in particular a reconstructed skin model.

For the purpose of the invention, the term “reconstructed skin model” isintended to denote a model in which various cell types, such as inparticular the natural constituents of the skin, like, for example,keratinocytes, fibroblasts, Langerhan cells and melanocytes, arecombined.

The fibroblast cells may or may not be irradiated.

Such models and the preparation thereof are known to those skilled inthe art.

Thus, the present invention is also directed towards a method ofpreparing an isolated reconstructed skin, comprising at least the stepconsisting in bringing at least one complex form according to theinvention into contact with cells capable of generating an isolatedreconstructed skin, and in particular keratinocytes.

The examples and figures which follow are presented by way ofnonlimiting illustration of the invention.

EXAMPLE 1

Development of a High-Sensitivity ELISA Assay for Assaying Complex Formsof CLSP in Accordance with the Invention:

CLSP plates developed, custom-made, with the MesoScale Discovery (MSD)technology were used in this assay. The monoclonal antibodies directedagainst a recombinant “full-length” CLSP protein fused with GST weredeveloped by the company

CovalAb (Lyon, France) (Méhul et al., 2006). The capture antibody (CloneDB15C9) was deposited at a concentration of 200 μg/ml on a standard96-well plate of “small spot” type

(MesoScale).

A reference curve (5 to 0.078 μg/ml) was established using therecombinant CLSP protein.

Each plate was blocked with a blocking buffer (MesoScale) for 1 hour atambient temperature. 25 μl of each sample and of standards weredeposited in triplicate and incubated with stirring for 1 hour atambient temperature. The plate was then washed 4 times with Tris buffer(MesoScale). The plates were then incubated with stirring for 1 /hour atambient temperature with 25 μl of detection antibody (Mesoscale)labelled with “sulpho-tag” (CLSP clone DB7g12) at a concentration of 1μg/ml. The plates were washed 4 times with a Tris buffer (MesoScale)before the addition of 150 μl of 1× read buffer T (MesoScale). Theplates were read on the “Sector Imager 6000” instrument. The data wasstandardized in terms of ng CLSP per μg of total protein.

EAMPLE 2

Characterization of the Content of CLSP Complex Forms in Various SamplesTaken by Stripping

This characterization is carried out by means of the ELISA assaydescribed in Example 1.

Two clinical studies were carried out: a knowledge study (2 groups of 20female individuals, with Caucasian skin: 30-35 years old and 60-65 yearsold) with samples taken from the forearm and the face, and a productstudy over 56 days, i.e. 2 months (1 group of 24 female individuals,with Caucasian skin, aged 40-45) with samples taken only from theforearm.

The product is a serum composed of 10% of bifidiobiotic (CLR complex)and 0.002% of phytosphingosine-SLC (treatment “G”).

The CLR complex relates to a lysate registered under the INCI name:Bifidat ferment Lysate, under the EINECS name: Bifidobacterium longum,under the EINECS No.: 306-168-4 and under CAS No.: 96507-89-0.

Such a lysate is in particular sold under the name Repair Complex CLR®by the company K. Richter GmbH.

The name Phytosphingosine-SLC corresponds to aphytosphingosine-salicylate derivative sold by the company EvonikGoldschmidt.

For both studies, D-squame samples were taken from the face (cheek) andfrom the forearm (posterior face). The D-squame corneodiscs sampled fromthe forearms were used to evaluate the product effects.

a) Extraction of the Proteins from the D-Squame Corneodiscs:

The corneodisc is placed in a 2 ml Eppendorf tube, sticky surface facinginwards, to which 550 pl of “Native+” extraction buffer (TBS, 1%TRITONTM X-100, 1M NaCl) and one stainless steel bread per tube areadded. The tube is then placed in the racks of the MM400 vibration mill(Retsch). The extraction is carried out by vibration milling for onecycle of 2 min at 30 Hz. The medium is then recovered and filteredthrough a 0.45 μm Millipore Ultrafree column, followed by centrifugationfor 5 min at 5000 g at 4° C. The supernatant is stored at −20° C. whilewaiting to be analysed.

b) Statistical Analysis

The analysis of the biomarkers for the age (young vs. aged) and region(arm vs. face) effects is carried out by means of a mixed model ofanalysis of variance with the age, region and age*region interactionfactors as fixed effects, and the subject factor as random effect. Theregion effect for an age category and the age effect for a region wererealized by means of contrasts.

The analysis of the biomarkers, for the treatment effect, is carried outby means of a mixed model of analysis of variance on the paired activeagent-placebo differences with the time factor as fixed effect and thesubject factor as random effect.

The treatment effect at D0 and D56 is tested by means of contrasts.

The variables were, when necessary, log-transformed beforehand in orderto make their distribution more Gaussian.

The SPSS software, version 14 is used for the descriptive analyses(graphs of the means and box plots), and the SAS Enterprise Guidesoftware, version 3 is used for the interference part.

The first-species alpha-risk was fixed at 5% in a two-sided approach.

The results of these two studies are given in FIGS. 1 and 2.

The data reveal a decrease in the complex forms of CLSP with age, asrepresented in FIG. 1.

The high-sensitivity ELISA kit that was developed indeed makes itpossible to verify that the expression of the complex forms of CLSPdecreases with age.

FIG. 2, for its part, represents the effect of the tested product on theamount of complex forms. A significant increase in the amount of complexforms is clearly observed, thereby demonstrating the activating effectof this product. The latter therefore provides a real anti-ageingeffect.

1. A method of screening for anti-ageing active agents, comprising: a)bringing at least one cell type containing at least one complex form ofcalmodulin-like skin protein, or CLSP, which derives at least in partfrom a polypeptide having an amino acid sequence encoded by a nucleicacid sequence represented completely or partly by SEQ ID NO 1 or ananalogue thereof, or a fragment thereof, into contact with at least onetest anti-ageing active agent, under conditions suitable for theexpression of said complex form, b) determining a content of saidcomplex form, and c) comparing said content determined in step b) with acontent of said complex form determined in the absence of test chemicalor biological compound.
 2. The method according to claim 1, wherein anincrease in complex form(s) of calmodulin-like skin protein, or CLSP,which derives at least in part from a polypeptide having an amino acidsequence encoded by a nucleic acid sequence represented completely orpartly by SEQ ID NO 1 or an analogue thereof, or a fragment thereof, isindicative an active agent with anti-ageing properties.
 3. A cosmeticmethod for characterizing the effectiveness of a cosmetic or therapeutictreatment intended to prevent and/or treat the signs of skin ageinglinked to chronological ageing in an individual, comprising at least thequalitative or quantitative characterization of at least one complexform of calmodulin-like skin protein, or CLSP, which derives at least inpart from a polypeptide having an amino acid sequence encoded by anucleic acid sequence represented completely or partly by SEQ ID NO 1 oran analogue thereof, or a fragment thereof.
 4. The method according toclaim 3, wherein an increase in complex form(s) of calmodulin-like skinprotein, or CLSP, which derives at least in part from a polypeptidehaving an amino acid sequence encoded by a nucleic acid sequencerepresented completely or partly by SEQ ID NO 1 or an analogue thereof,or a fragment thereof, is indicative of said treatment of the signs ofskin ageing being effective.
 5. The method according to claim 3, furthercomprising at least one step consisting in administering, to saidindividual, a cosmetic care composition established or selected withregard to the information obtained at the end of the characterizationstep.
 6. A nontherapeutic method for characterizing the effectiveness ofa treatment capable of preventing and/or treating the signs of skinageing in an individual, comprising: i. providing at least a first skinsurface sample representative of said individual, ii. quantifying, insaid sample, especially via an ELISA assay technique, at least onecomplex form of calmodulin-like skin protein, or CLSP, which derives atleast in part from a polypeptide having an amino acid sequence encodedby a nucleic acid sequence represented completely or partly by SEQ ID NO1 or an analogue thereof, or a fragment thereof, iii. repeating steps i.and ii. on a second skin surface sample representative of saidindividual, and iv. comparing the results obtained at the end of stepsii. and iii., in particular in order to deduce therefrom informationrelating to at least one effect of said treatment, said first and secondskin surface samples corresponding to different treatment stages.
 7. Themethod according to claim 6, wherein the first sample is representativeof a pre-treatment state and the second is representative of a stateduring the course of treatment or a post-treatment state.
 8. The methodaccording to claim 6, wherein an increase in complex form(s) isindicative of said treatment of the signs of skin ageing beingeffective.
 9. The method according to claim 6, further comprising atleast one step consisting in administering, to said individual, acosmetic care composition established or selected with regard to theinformation obtained at the end of step iv.
 10. A method for screeningfor biological or chemical compound capable of modulating the increasein and/or the stability and/or the biological activity of at least onecomplex form of calmodulin-like skin protein, or CLSP, which derives atleast in part from a polypeptide having an amino acid sequence encodedby a nucleic acid sequence represented completely or partly by SEQ ID NO1 or an analogue thereof, or a fragment thereof, comprising contactingat least said complex form with at least one biological or chemicalcompound to be screened.